Currently available ELISAs for paratuberculosis employ a preadsorption step with Mycobacterium phlei. As M. fortuitum is the most frequently isolated environmental mycobacteria in Brazil, we considered the hypothesis that the use of local strains of environmental mycobacteria might be useful. Ten negative sera and four positive sera of our collection, confirmed by results of bacteriological culture, plus one positive and one negative control serum were used. Adsorption of bovine sera was performed in three distinct ways: using M.phlei only, M.fortuitum only or a combination of M. phlei + M. fortuitum. In spite of the overall reduction on ODs values observed at the M. fortuitum-ELISA, three positive sera remained presenting much higher values (mean = 0.650) than negative sera (mean= 0.150), as expected. Only one positive serum became negative with an OD value of 0.261 (cut-off = 0.35). Two of the positive sera and four of the negative sera preadsorbed with the M. fortuitum + M. phlei solution presented higher ODs than with the standard assay. Nevertheless, with this preadsorption step, no serum changed its final status and correlation between both tests was also high for those samples (k> 0.8). In spite of one serum having its final result altered, the assays using different preadsorptions were demonstrated to be comparable (p<0.01) and no difference on efficacy could be detected between them (k>0.8). Besides of this, variation on ODs values observed among the three preadsorption assays was not significant (p< 0.01). Our results suggest that M. fortuitum, alone or combined with M. phlei, may be considered as an alternative for the preadsorption step of ELISAs for paratuberculosis.